Proteases from gram-positive organisms

ABSTRACT

The present invention relates to the identification of novel metallo-proteases (MP) in Gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for  Bacillus  (MP). The present invention also provides host cells having mutation or deletion of part or all of the gene encoding MP. The present invention also provides host cells further comprising nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides cleaning compositions comprising an MP of the present invention.

FIELD OF THE INVENTION

The present invention relates to metallo-proteases derived fromgram-positive microorganisms. The present invention provides nucleicacid and amino acid sequences of a metallo-protease identified inBacillus subtilis. The present invention also provides methods for theproduction of the protease in host cells as well as the production ofheterologous proteins in a host cell having a mutation or deletion ofpart or all of the proteases of the present invention.

BACKGROUND OF THE INVENTION

Gram-positive microorganisms such as members of the group Bacillus, havebeen used for large-scale industrial fermentation due, in part, to theirability to secrete their fermentation products into the culture media.In gram-positive bacteria, secreted proteins are exported across a cellmembrane and a cell wall, and then are subsequently released into theexternal media usually maintaining their native conformation.

Various gram-positive microorganisms are known to secrete extracellularand/or intracellular protease at some stage in their life cycles. Manyproteases are produced in large quantities for industrial purposes. Anegative aspect of the presence of proteases in gram-positive organismsis their contribution to the overall degradation of secretedheterologous or foreign proteins.

The classification of proteases found in microorganisms is based ontheir catalytic mechanism which results in four groups: the serineproteases; metallo-proteases; cysteine proteases; and asparticproteases. These categories, in general, can be distinguished by theirsensitivity to various inhibitors. For example, the serine proteases areinhibited by phenylmethylsulfonylfluoride (PMSF) anddiisopropylfluorophosphate (DIFP); the metallo-proteases by chelatingagents; the cysteine enzymes by iodoacetamide and heavy metals and theaspartic proteases by pepstatin. The serine proteases have alkaline pHoptima, the metalloproteases are optimally active around neutrality, andthe cysteine and aspartic enzymes have acidic pH optima (BiotechnologyHandbooks. Bacillus. vol. 2, edited by Harwood, 1989 Plenum Press, NewYork).

Metallo-proteases form the most diverse of the catalytic types ofproteases. Family M23 contains bacterial enzymes such as the β-lyticendopeptidases of Lysobacter and Achromobacter and the Pseudomonas LasAprotein and have specificity for Gly bonds, especially inGly-Gly+Xaa-sequences (Methods in Enzymology, vol. 248, Academic Press,Inc. 1994). The enzymes of the M23 family contain zinc and a conservedHis-Xaa-His motif

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a heretofore unknownmetallo-protease (MT) found in gram positive microorganisms, uses of theMP in industrial applications, and advantageous strain improvementsbased on genetically engineering such microorganisms to delete,underexpress or overexpress that MP. Due to the overall relatedness ofMP with Pseudomonas lasA protein, including the presence of the motifHis-Xaa-His, MP appears to be a member of the metallo-protease familyM23.

Applicant's discovery, in addition to providing a new and usefulprotease and methods of detecting DNA encoding such proteases in a grampositive microorganism, provides several advantages which may facilitateoptimization and/or modification of strains of gram positivemicroorganisms, such as Bacillus, for expression of desired, e.g.heterologous, proteins. Such optimizations, as described below indetail, allow the construction of strains having decreased proteolyticdegradation of desired expression products.

Applicant's invention is further based on the discovery of the presenceof MP's in Gram-positive microorganisms. The Gram-positive microorganismmay be Bacillus and may also be selected from the group consisting ofBacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformisand Bacillus amyloliquifaciens. The present invention further relies onthe discovery that naturally occurring MP is encoded by nucleic acidfound about 2248 kb from the point of origin of Bacillus subtilis I-168strain (Bacillus Genetic Stock Center, accession number 1A1, Columbus,Ohio). The present invention relates to the MP encoded thereby, as wellas the nucleic acid and amino acid molecules having the sequencesdisclosed in FIGS. 1A-1O.

The present invention thus provides methods for detecting gram positivemicroorganism homologs of B. subtilis MP that comprises hybridizing partor all of the nucleic acid encoding B. subtilis MP with nucleic acidderived from gram-positive organisms, either of genomic or cDNA origin.Accordingly, the present invention provides a method for detecting agram-positive microorganism MP, comprising the steps of hybridizinggram-positive microorganism nucleic acid under low stringency conditionsto a probe, wherein the probe comprises part or all of the nucleic acidsequence shown in FIGS. 1A-1O; and isolating gram-positive nucleic acidwhich hybridizes to said probe.

In a preferred embodiment, the Bacillus is selected from the groupconsisting of B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus and B. thuringiensis.

The production of desired heterologous proteins or polypeptides ingram-positive microorganisms may be hindered by the presence of one ormore proteases, including MP, which degrade the produced heterologousprotein or polypeptide. One advantage of the present invention is thatit provides methods and expression systems which can be used to preventthat degradation, thereby enhancing yields of the desired heterologousprotein or polypeptide. Accordingly, the present invention provides agram-positive microorganism having a mutation or deletion of part or allof the gene encoding MP, which results in the inactivation of the MPproteolytic activity, either alone or in combination with mutations inother proteases, such as apr, npr, epr, mpr, bpf or isp for example, orother proteases known to those of skill in the art. In one embodiment ofthe present invention, the gram-positive organism is a member of thegenus Bacillus. In another embodiment, the Bacillus is selected from thegroup consisting of B. subtilis, B. licheniformis, B. lenius, B. brevis,B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus and Bacillus thuringiensis. In afurther preferred embodiment, the Bacillus is Bacillus subtilis.

In another aspect, the gram-positive host having one or moremetallo-protease deletions or mutations is further geneticallyengineered to produce a desired protein. In one embodiment of thepresent invention, the desired protein is heterologous to thegram-positive host cell. In another embodiment, the desired protein ishomologous to the host cell. The present invention encompasses agram-positive host cell having a deletion, mutation or interruption ofthe nucleic acid encoding the naturally occurring homologous protein,such as a protease, and having nucleic acid encoding the homologousprotein re-introduced in a recombinant form. In another embodiment, thehost cell produces the homologous protein. Accordingly, the presentinvention also provides methods and expression systems for reducingdegradation of heterologous proteins produced in gram-positivemicroorganisms. The gram-positive microorganism may be normallysporulating or non-sporulating. In a preferred embodiment, the grampositive host cell is a Bacillus. In another preferred embodiment, theBacillus host cell is Bacillus. In another embodiment, the Bacillus isselected from the group consisting of B. subtilis, B. licheniformis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillusthuringiensis.

Naturally occurring gram positive MP as well as proteolytically activeamino acid variations or derivatives thereof, have application in thetextile industry, in cleaning compositions and in animal feed. Themetallo-protease MP may be used alone or in combination with otherenzymes and/or mediators or enhancers. Accordingly, in a further aspectof the present invention, gram-positive MP is produced on an industrialfermentation scale in a microbial host expression system. The presentinvention provides a cleaning to composition comprising ametalloprotease, MP, having the amino acid sequence shown in FIGS. 1A-1Oor the amino acid encoded by the MP nucleic acid found at about 2248kilobases from the point of origin of Bacillus subtilis. Also providedare cleaning compositions comprising a metalloprotease having at least80%, at least 90%, or at least 95% homology with the amino acid sequenceshown in FIGS. 1A-1O or comprising a metalloprotease encoded by a genethat hybridizes with the nucleic acid shown in FIGS. 1A-1O under highstringency conditions.

Further there is provided an animal feed comprising a metalloprotease,MP, having the amino acid sequence shown in FIGS. 1A-1O. Also providedare animal feeds comprising a metalloprotease having at least 80%, atleast 90%, and at least 95% homology with the amino acid sequence shownin FIGS. 1A-1O or comprising a metalloprotease encoded by a gene thathybridizes with the nucleic acid shown in FIGS. 1A-1O under highstringency conditions.

Also provided is a composition for the treatment of a textile comprisinga metalloprotease, MP, having the amino acid sequence shown in FIGS.1A-1O. Also provided are compositions for the treatment of a textilecomprising a metalloprotease having at least 80%, at least 90%, or atleast 95% homology with the amino acid sequence shown in FIGS. 1A-1O orcomprising a metalloprotease encoded by a gene that hybridizes with thenucleic acid shown in FIGS. 1A-1O under high stingency conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1O shows the DNA and amino acid sequence for Bacillus subtilisMP.

FIG. 2 show an amino acid alignment of Bacillus subtilis MP (designatedas YOMI) and Pseudomonas LasA. The amino acid motif H-X-H is noted atamino acid 308-310 in LasA

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

As used herein, the genus Bacillus includes all members known to thoseof skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B. lautusand B. thuringiensis.

The present invention relates to a newly characterized metallo-protease(MP) from gram positive organisms. In a preferred embodiment, themetallo-protease is obtainable from a gram-positive organism which is aBacillus. In another preferred embodiment, the metallo-protease isobtainable from a Bacillus which is selected from the group consistingof B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. ciculans, B. lautus and B. thuringiensis.

In another preferred embodiment, the gram-positive organism is Bacillussubtilis and MP has the amino acid sequence encoded by the nucleic acidmolecule having the sequence that occurs around 2248 kilobases from thepoint of origin of Bacillus subtilis I-168.

In another preferred embodiment, Bacillus subtilis has the nucleic acidand amino acid sequence as shown in FIGS. 1A-1O. The present inventionencompasses the use of amino acid variations of the amino acid sequencesdisclosed in FIGS. 1A-1O that have proteolytic activity. Suchproteolytic amino acid variants can be used in the textile industry,animal feed and in cleaning compositions. The present invention alsoencompasses the use of B. subtilis amino acid variations or derivativesthat are not proteolytically active. DNA encoding such variants can beused in methods designed to delete or mutate the naturally occurringhost cell MP.

As used herein, “nucleic acid” refers to a nucleotide or polynucleotidesequence, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof A “polynucleotide homolog” as used herein refers to agram-positive microorganism polynucleotide that has at least 80%, atleast 90% and at least 95% identity to B. subtilis MP, or which iscapable of hybridizing to B. subtilis MP under conditions of highstringency and which encodes an amino acid sequence havingmetallo-protease activity.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or amino acid that is removed from at least one component withwhich it is naturally associated.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in a gram-positive host cell.Examples of heterologous proteins include enzymes such as hydrolasesincluding proteases; cellulases, amylases, carbohydrases, and lipases;isomerases such as racemases, epimerases, tautomerases, or mutases;transferases, kinases and phophatases. The heterologous gene may encodetherapeutically significant proteins or peptides, such as growthfactors, cytokines, ligands, receptors and inhibitors, as well asvaccines and antibodies. The gene may encode commercially importantindustrial proteins or peptides, such as proteases, carbohydrases suchas amylases and glucoamylases, cellulases, oxidases and lipases. Thegene of interest may be a naturally occurring gene, a mutated gene or asynthetic gene.

The term “homologous protein” refers to a protein or polypeptide nativeor naturally occurring in a gram-positive host cell. The inventionincludes host cells producing the homologous protein via recombinant DNAtechnology. The present invention encompasses a gram-positive host cellhaving a deletion or interruption of the nucleic acid encoding thenaturally occurring homologous protein, such as a protease, and havingnucleic acid encoding the homologous protein re-introduced in arecombinant form. In another embodiment, the host cell produces thehomologous protein.

As used herein, the term “overexpressing” when referring to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than its production in its naturallyoccurring environment.

As used herein, the phrase “proteolytic activity” refers to a proteinthat is able to hydrolyze a peptide bond. Enzymes having proteolyticactivity are described in Enzyme Nomenclature, 1992, edited WebbAcademic Press, Inc.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The unexpected discovery of the metallo-protease M23 family member,designated herein as MP, found in translated, uncharacterized B.subtilis genomic sequences provides a basis for producing host cells,expression methods and systems which can be used to prevent thedegradation of recombinantly produced heterologous proteins.

Accordingly, in a preferred embodiment, the host cell is a gram-positivehost cell that has a deletion or mutation in the naturally occurringnucleic acid encoding MP said mutation resulting in deletion orinactivation of the production by the host cell of the MN proteolyticgene product. The host cell may additionally be genetically engineeredto produced a desired protein or polypeptide.

It may also be desired to genetically engineer host cells of any type toproduce a gram-positive MP. Such host cells are used in large scalefermentation to produce large quantities of the protease which may beisolated or purified and used in cleaning products, such as detergents,in textile treatments and as animal feed additives.

I. MP Sequences

The nucleic acid sequence and amino acid sequence for Bacillus subtilisMP are shown in FIGS. 1A-1O. As will be understood by the skilledartisan, due to the degeneracy of the genetic code, a variety ofpolynucleotides can encode the Bacillus subtilis MP having the aminoacid sequence shown in FIGS. 1A-1O. The present invention encompassesall such polynucleotides.

The present invention encompasses the use of MP polynucleotide homologsencoding gram-positive microorganism MPs which have at least 80%, or atleast 90% or at least 95% identity to B. subtilis MP shown in FIGS.1A-1O as long as the homolog encodes a protein that has proteolyticactivity.

Gram-positive polynucleotide homologs of B. subtilis MP may be obtainedby standard procedures known in the art from, for example, cloned DNA(e.g., a DNA “library”), genomic DNA libraries, by chemical synthesisonce identified, by cDNA cloning, or by the cloning of genomic DNA, orfragments thereof, purified from a desired cell. (See, for example,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II.) A preferred source is from genomic DNA.

As will be understood by those of skill in the art, the polynucleotidesequence disclosed in FIGS. 1A-1O may reflect inadvertent errorsinherent to nucleic acid sequencing technology. Moreover, the sequenceof polynucleotides derived from related species, e.g., other Bacillus,will contain variations to the sequences specifically disclosed herein.Nonetheless, one of ordinary skill in the art is fully capable ofdetermining the correct sequences from the information provided hereinregarding the invention. For example, as described below, it is possibleto identify the MP of the invention by virtue of its location in themicroorganism's genome. The present invention encompasses the naturallyoccurring nucleic acid molecule having the nucleic acid, sequenceobtained from the genomic sequence of Bacillus species.

Nucleic acid encoding Bacillus subtilis MP starts around 2248 kilobasescounting from the point of origin in the Bacillus subtilis strain I-168(Anagnostopala, 1961, J. Bacteriol. 81:741-746 or Bacillus Genomic StockCenter, accession 1A1, Columbus, Ohio). The Bacillus subtilis point oforigin has been described in Ogasawara, N. (1995, Microbiology 141:Pt.2257-59). Bacillus subtilis MP has a length of 2285 amino acids. Basedupon the location of the DNA encoding Bacillus subtilis MP, naturallyoccurring B. subtilis MP can be obtained by methods known to those ofskill in the art including PCR technology.

Oligonucleotide sequences or primers of about 10-30 nucleotides inlength can be designed from the polynucleotide sequence disclosed inFIGS. 1A-1O and used in PCR technology to isolate the naturallyoccurring sequence from B. subtilis genomic sequences.

Another general strategy for the “cloning” of B. subtilis genomic DNApieces for sequencing uses inverse PCR. A known region is scanned for aset of appropriate restriction enzyme cleavage sites and inverse PCR isperformed with a set of DNA primers determined from the outermost DNAsequence. The DNA fragments from the inverse PCR are directly used astemplate in the sequencing reaction. The newly derived sequences can beused to design new oligonucleotides. These new oligonucleotides are usedto amplify DNA fragments with genomic DNA as template. The sequencedetermination on both strands of a DNA region is finished by applying aprimer walking strategy on the genomic PCR fragments. The benefit ofmultiple starting points in the primer walking results from the seriesof inverse PCR fragments with different sizes of new “cloned” DNApieces. From the most external DNA sequence a new round of inverse PCRis started. The whole inverse PCR strategy is based on the sequentialuse of conventional taq polymerase and the use of long range inverse PCRin those cases in which the taq polymerase failed to amplify DNAfragments. Nucleic acid sequencing is performed using standardtechnology. One method for nucleic acid sequencing involves the use of aPerkin-Elmer Applied Biosystems 373 DNA sequencer (Perkin-Elmer, FosterCity, Calif.).

Nucleic acid sequences derived from genomic DNA may contain regulatoryregions in addition to coding regions. Whatever the source, the isolatedMP gene should be molecularly cloned into a suitable vector forpropagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the MP may be accomplished in a number of ways. Forexample, a B. subtilis MP gene of the present invention, or its specificRNA, or a fragment thereof, such as a probe or primer, may be isolatedand labeled and then used in hybridization assays to detect agram-positive MP gene. (Benton, W. and Davis, R., 1977, Science 196:180;Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. USA72:3961). Those DNA fragments sharing substantial sequence similarity tothe probe will hybridize under stringent conditions.

Accordingly, the present invention provides a method for the detectionof gram-positive MP polynucleotide homologs which comprises hybridizingpart or all of a nucleic acid sequence of B. subtilis MP withgram-positive microorganism nucleic acid of either genomic or cDNAorigin.

Also included within the scope of the present invention is the use ofgram-positive microorganism polynucleotide sequences that are capable ofhybridizing to the nucleotide sequence of B. subtilis MP underconditions of intermediate to maximal stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex, as taught in Berger and Kimmel (1987, Guide toMolecular Cloning Techniques, Methods in Enzymology. Vol 152. AcademicPress, San Diego Calif.) incorporated herein by reference and confer adefined “stringency” as explained below.

“Maximum stringency” typically occurs at about Tm-5° C. (5° C. below theTm of the probe); “high stringency” at about 5° C. to 10° C. below Tm;“intermediate stringency” at about 10° C. to 20° C. below Tm; and “lowstringency” at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate or low stringency hybridization can be used to identify ordetect polynucleotide sequence homologs.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.).

The process of amplification as carried out in polymerase chain reaction(PCR) technologies is described in Dieffenbach C W and G S Dveksler(1995, PCR Primer, a

Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.). A nucleicacid sequence of at least about 10 nucleotides and as many as about 60nucleotides from B. subtilis MP preferably about 12 to 30 nucleotides,and more preferably about 20-25 nucleotides can be used as a probe orPCR primer.

The B. subtilis MP amino acid sequences (shown in FIGS. 1A-1O) wereidentified via a BLAST search (Altschul, Stephen, Basic local alignmentsearch tool, J. Mol. Biol. 215:403-410) of Bacillus subtilis genomicnucleic acid sequences. B. subtilis MP (YOMI) was identified by itsoverall nucleic acid identity to the metallo-protease, Pseudomonas lasA,including the presence of the catalytic domain H-X-H as shown in FIG. 2.

II. Expression Systems

The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of desiredheterologous or homologous proteins in gram-positive microorganisms. Inone embodiment, a host cell is genetically engineered to have a deletionor mutation in the gene encoding a gram-positive MP such that therespective activity is deleted. In another embodiment of the presentinvention, a gram-positive microorganism is genetically engineered toproduce a metallo-protease of the present invention.

Inactivation of a Gram-Positive Metallo-Protease in a Host Cell

Producing an expression host cell incapable of producing the naturallyoccurring metallo-protease necessitates the replacement and/orinactivation of the naturally occurring gene from the genome of the hostcell. In a preferred embodiment, the mutation is a non-revertingmutation.

One method for mutating nucleic acid encoding a gram-positivemetallo-protease is to clone the nucleic acid or part thereof, modifythe nucleic acid by site directed mutagenesis and reintroduce themutated nucleic acid into the cell on a plasmid. By homologousrecombination, the mutated gene may be introduced into the chromosome.In the parent host cell, the result is that the naturally occurringnucleic acid and the mutated nucleic acid are located in tandem on thechromosome. After a second recombination, the modified sequence is leftin the chromosome having thereby effectively introduced the mutationinto the chromosomal gene for progeny of the parent host cell.

Another method for inactivating the metallo-protease proteolyticactivity is through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoded metallo-protease gene. In another preferred embodiment,nucleic acid encoding the catalytic amino acid residues are deleted.

Deletion of the naturally occurring gram-positive microorganismmetallo-protease can be carried out as follows. A metallo-protease geneincluding its 5′ and 3′ regions is isolated and inserted into a cloningvector. The coding region of the metallo-protease gene is deleted formthe vector in vitro, leaving behind a sufficient amount of the 5′ and 3′flanking sequences to provide for homologous recombination with thenaturally occurring gene in the parent host cell. The vector is thentransformed into the gram-positive host cell. The vector integrates intothe chromosome via homologous recombination in the flanking regions.This method leads to a gram-positive strain in which the protease genehas been deleted.

The vector used in an integration method is preferably a plasmid. Aselectable marker may be included to allow for ease of identification ofdesired recombinant microorgansims. Additionally, as will be appreciatedby one of skill in the art, the vector is preferably one which can beselectively integrated into the chromosome. This can be achieved byintroducing an inducible origin of replication, for example, atemperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

Integration by the Campbell-type mechanism can take place in the 5′flanking region of the protease gene, resulting in a protease positivestrain carrying the entire plasmid vector in the chromosome in themetallo-protease locus. Since illegitimate recombination will givedifferent results it will be necessary to determine whether the completegene has been deleted, such as through nucleic acid sequencing orrestriction maps.

Another method of inactivating the naturally occurring metallo-proteasegene is to mutagenize the chromosomal gene copy by transforming agram-positive microorganism with oligonucleotides which are mutagenic.Alternatively, the chromosomal metallo-protease gene can be replacedwith a mutant gene by homologous recombination.

The present invention encompasses host cells having additional proteasedeletions or mutations, such as deletions or mutations in apr, npr, epr,mpr and others known to those of skill in the art.

One assay for the detection of mutants involves growing the Bacillushost cell on medium containing a protease substrate and measuring theappearance or lack thereof, of a zone do clearing or halo around thecolonies. Host cells which have an inactive protease will exhibit littleor no halo around the colonies.

III. Production of Metallo-Protease

For production of metallo-protease in a host cell, an expression vectorcomprising at least one copy of nucleic acid encoding a gram-positivemicroorganism MP, and preferably comprising multiple copies, istransformed into the host cell under conditions suitable for expressionof the metallo-protease. In accordance with the present invention,polynucleotides which encode a gram-positive microorganism MP, orfragments thereof, or fusion proteins or polynucleotide homologsequences that encode amino acid variants of B. subtilis MP, may be usedto generate recombinant DNA molecules that direct their expression inhost cells. In a preferred embodiment, the gram-positive host cellbelongs to the genus Bacillus. In another preferred embodiment, the grampositive host cell is B. subtilis.

As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulargram-positive host cell (Murray E et al (1989) Nuc Acids Res 17:477-508)can be selected, for example, to increase the rate of expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

Altered MP polynucleotide sequences which may be used in accordance withthe invention include deletions, insertions or substitutions ofdifferent nucleotide residues resulting in a polynucleotide that encodesthe same or a functionally equivalent MP homolog, respectively. As usedherein a “deletion” is defined as a change in either nucleotide or aminoacid sequence in which one or more nucleotides or amino acid residues,respectively, are absent.

As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid residues, respectively, ascompared to the naturally occurring MP.

As used herein “substitution” results from the replacement of one ormore nucleotides or amino acids by different nucleotides or amino acids,respectively.

The encoded protein may also show deletions, insertions or substitutionsof amino acid residues which produce a silent change and result in afunctionally equivalent MP variant. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the variant retains the ability to modulatesecretion. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine;glycine, alanine; asparagine, glutamine; serine, threonine,phenylalanine, and tyrosine.

The MP polynucleotides of the present invention may be engineered inorder to modify the cloning, processing and/or expression of the geneproduct. For example, mutations may be introduced using techniques whichare well known in the art, eg, site-directed mutagenesis to insert newrestriction sites, to alter glycosylation patterns or to change codonpreference, for example.

In one embodiment of the present invention, a gram-positivemicroorganism MP polynucleotide may be ligated to a heterologoussequence to encode a fusion protein. A fusion protein may also beengineered to contain a cleavage site located between themetallo-protease nucleotide sequence and the heterologous proteinsequence, so that the metallo-protease may be cleaved and purified awayfrom the heterologous moiety.

IV. Vector Sequences

Expression vectors used in expressing the metallo-proteases of thepresent invention in gram-positive microorganisms comprise at least onepromoter associated with a metallo-protease selected from the groupconsisting of MP, which promoter is functional in the host cell. In oneembodiment of the present invention, the promoter is the wild-typepromoter for the selected metallo-protease and in another embodiment ofthe present invention, the promoter is heterologous to themetallo-protease, but still functional in the host cell. In onepreferred embodiment of the present invention, nucleic acid encoding themetallo-protease is stably integrated into the microorganism genome.

In a preferred embodiment, the expression vector contains a multiplecloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in thegram-positive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

V. Transformation

A variety of host cells can be used for the production Bacillus subtilisMP or MP homologs including bacterial, fungal, mammalian and insectscells. General transformation procedures are taught in Current ProtocolsIn Molecular Biology (vol. 1, edited by Ausubel et al., John Wiley &Sons, Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 95/14099, published26 May 1995).

In a preferred embodiment, the host cell is a gram-positivemicroorganism and in another preferred embodiment, the host cell isBacillus. In one embodiment of the present invention, nucleic acidencoding one or more MP(s) of the present invention is introduced into ahost cell via an expression vector capable of replicating within theBacillus host cell. Suitable replicating plasmids for Bacillus aredescribed in Molecular Biological Methods for Bacillus, Ed. Harwood andCutting, John Wiley & Sons, 1990, hereby expressly incorporated byreference; see chapter 3 on plasmids. Suitable replicating plasmids forB. subtilis are listed on page 92.

In another embodiment, where it is desired to produce the MP for use incleaning compositions, nucleic acid encoding MP is stably integratedinto the microorganism genome. Preferred host cells are gram-positivehost cells. Another preferred host is Bacillus. Another preferred hostis Bacillus subtilis. Several strategies have been described in theliterature for the direct cloning of DNA in Bacillus. Plasmid markerrescue transformation involves the uptake of a donor plasmid bycompetent cells carrying a partially homologous resident plasmid(Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen.Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol.154(3):1077-1087 (1983); and Weinrauch et al., J. Bacteriol.169(3):1205-1211 (1987)). The incoming donor plasmid recombines with thehomologous region of the resident “helper” plasmid in a process thatmimics chromosomal transformation.

Protoplast transformation is described for B. subtilis in Chang andCohen, (1979) Mol. Gen. Genet 168:111-115; for B. megaterium inVorobjeva et al., (1980) FEMS Microbiol. Letters 7:261-263; for B.amyloliquefaciens in Smith et al., (1986) Appl. and Env. Microbiol.51:634, for B. thuringiensis in Fisher et al., (1981) Arch. Microbiol.139:213-217; for B. sphaericus in McDonald (1984) J. Gen. Microbiol.130'203; and B. larvae in Bakhiet et al., (1985, Appi. Environ.Microbiol. 49:577). Mann et al., (1986, Current Microbiol. 13:131-135)report on transformation of Bacillus protoplasts and Holubova, (1985)Folia Microbiol. 30:97) disclose methods for introducing DNA intoprotoplasts using DNA containing liposomes.

VI. Identification of Transformants

Whether a host cell has been transformed with a mutated or a naturallyoccurring gene encoding a gram-positive MP, detection of thepresence/absence of market gene expression can suggest whether the geneof interest is present However, its expression should be confirmed. Forexample, if the nucleic acid encoding an MP of the present invention isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the MP under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicatesexpression of the MP as well.

Alternatively, host cells which contain the coding sequence for ametallo-protease and express the protein may be identified by a varietyof procedures known to those of skill in the art. These proceduresinclude, but are not limited to, DNA-DNA or DNA-RNA hybridization andprotein bioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

The presence of the metallo-protease polynucleotide sequence can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of B. subtilis MP.

VII. Assay of Protease Activity

There are various assays known to those of skill in the art fordetecting and measuring protease activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or colorimetrically using the Folin method(Bergmeyer, et al., 1984, Methods of Enzymatic Analysis vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology (W. M,Fogarty, ed.), Applied Science, London, pp. 251-317).

VIII. Secretion of Recombinant Proteins

Means for determining the levels of secretion of a heterologous orhomologous protein in a gram-positive host cell and detecting secretedproteins include, using either polyclonal or monoclonal antibodiesspecific for the protein. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA) and fluorescent activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton R et al (1990, Serological Methods, a LaboratoryManual, APS Press, St Paul Minn.) and Maddox D E et al (1983, J Exp Med158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the is art and can be used in various nucleic and amino acidassays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 and incorporated herein by reference.

IX. Purification of Proteins

Gram positive host cells transformed with polynucleotide sequencesencoding heterologous or homologous protein may be cultured underconditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinantgram-positive host cell comprising a mutation or deletion of themetallo-protease activity will be secreted into the culture media. Otherrecombinant constructions may join the heterologous or homologouspolynucleotide sequences to nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins (Kroll D Jet al (1993) DNA Cell Biol 12:441-53).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath J (1992) Protein Expr Purif3:263-281), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego Calif.) between the purification domain and the heterologousprotein can be used to facilitate purification.

X. Uses of The Present Invention MP and Genetically Engineered HostCells

The present invention provides genetically engineered host cellscomprising mutations, preferably non-revertable mutations, or deletionsin the naturally occurring gene encoding MP such that the proteolyticactivity is diminished or deleted altogether. The host cell may containadditional protease deletions, such as deletions of the mature subtilisnprotease and/or mature neutral protease disclosed in U.S. Pat. No.5,264,366.

In a preferred embodiment, the host cell is further geneticallyengineered to produce a desired protein or polypeptide. In a preferredembodiment the host cell is a Bacillus. In another preferred embodiment,the host cell is a Bacillus subtilis.

In an alternative embodiment, a host cell is genetically engineered toproduce a gram-positive MP. In a preferred embodiment, the host cell isgrown under large scale fermentation conditions. In another preferredembodiment, the MP is isolated and/or purified and used in the textileindustry, the feed industry and in cleaning compositions such asdetergents.

As noted, MP can be useful in formulating various cleaning compositions.A number of known compounds are suitable surfactants useful incompositions comprising the MP of the invention. These include nonionic,anionic, cationic, anionic or zwitterionic detergents, as disclosed inU.S. Pat. No. 4,404,128 and U.S. Pat. No. 4,261,868. A suitabledetergent formulation is that described in Example 7 of U.S. Pat. No.5,204,015. The art is familiar with the different formulations which canbe used as cleaning compositions. In addition, MP can be used, forexample, in bar or liquid soap applications, dishcare formulations,contact lens cleaning solutions or products, peptide hydrolysis, wastetreatment, textile applications, as fusion-cleavage enzymes in proteinproduction, etc. MP may comprise enhanced performance in a detergentcomposition (as compared to another detergent protease). As used herein,enhanced performance in a detergent is defined as increasing cleaning ofcertain enzyme sensitive stains such as grass or blood, as determined byusual evaluation after a standard wash cycle.

MP can be formulated into known powdered and liquid detergents having pHbetween 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably0.1% to 0.5%) by weight. These detergent cleaning compositions can alsoinclude other enzymes such as known proteases, amylases, cellulases,lipases or endoglycosidases, as well as builders and stabilizers.

The addition of MP to conventional cleaning compositions does not createany special use limitation. In other words, any temperature and pHsuitable for the detergent is also suitable for the present compositionsas long as the pH is within the above range, and the temperature isbelow the described MP's denaturing temperature. In addition, MP can beused in a cleaning composition without detergents, again either alone orin combination with builders and stabilizers.

Proteases can be included in animal feed such as part of animal feedadditives as described in, for example, U.S. Pat. No. 5,612,055; U.S.Pat. No. 5,314,692; and U.S. Pat. No. 5,147,642.

One aspect of the invention is a composition for the treatment of atextile that includes MP. The composition can be used to treat forexample silk or wool as described in publications such as RD 216,034; EP134,267; U.S. Pat. No. 4,533,359; and EP344,259.

MP Polynucleotides

A B. subtlis MP polynucleotide, or any part thereof, provides the basisfor detecting the presence of gram-positive microorganism MPpolynucleotide homologs through hybridization techniques and PCRtechnology.

Accordingly, one aspect of the present invention is to provide fornucleic acid hybridization and PCR probes which can be used to detectpolynucleotide sequences, including genomic and cDNA sequences, encodinggram-positive MP or portions thereof. In another aspect of the presentinvention, an MP polynucleotide can be used in hybridization technologyto detect the major protease of a gram-positive microorganism due to theproximity of the MP with the major protease.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto

EXAMPLE I Preparation of a Genomic Library

The following example illustrates the preparation of a Bacillus genomiclibrary.

Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc. 1987, chapter 2. 4.1., Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genomic DNA is recovered from the resultingsupernatant by isopropanol precipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

After obtaining purified genomic DNA, the DNA is subjected to Sau3Adigestion. Sau3A recognizes the 4 base pair site GATC and generatesfragments compatible with several convenient phage lambda and cosmidvectors. The DNA is subjected to partial digestion to increase thechance of obtaining random fragments.

The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II Detection of Gram-Positive Microorganisms

The following example describes the detection of gram-positivemicroorganism MP.

DNA derived from a gram-positive microorganism is prepared according tothe methods disclosed in Current Protocols in Molecular Biology, Chap. 2or 3. The nucleic acid is subjected to hybridization and/or PCRamplification with a probe or primer derived from MP.

The nucleic acid probe is labeled by combining 50 pmol of the nucleicacid and 250 mCi of [gamma ³²P] adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®, Boston Mass.).The labeled probe is purified with Sephadex G-25 super fine resin column(Pharmacia). A portion containing 10⁷ counts per minute of each is usedin a typical membrane based hybridization analysis of nucleic acidsample of either genomic or cDNA origin.

The DNA sample which has been subjected to restriction endonucleasedigestion is fractionated on a 0.7 percent agarose gel and transferredto nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.).Hybridization is carried out for 16 hours at 40 degrees C. To removenonspecific signals, blots are sequentially washed at room temperatureunder increasingly stringent conditions up to 0.1× saline sodium citrateand 0.5% sodium dodecyl sulfate. The blots are exposed to film forseveral hours, the film developed and hybridization patterns arecompared visually to detect polynucleotide homologs of B. subtilis MP.The homologs are subjected to confirmatory nucleic acid sequencing.Methods for nucleic acid is sequencing are well known in the art.Conventional enzymatic methods employ DNA polymerase Kienow fragment,SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest.

Various other examples and modifications of the foregoing descriptionand examples will be apparent to a person skilled in the art afterreading the disclosure without departing from the spirit and scope ofthe invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1-10. (Cancelled)
 11. A cleaning composition comprising a gram positivemicroorganism metalloprotease (MP).
 12. The cleaning composition ofclaim 11 wherein the gram positive microorganism is a Bacillus.
 13. Thecleaning composition of claim 11 wherein the MP has the amino acidsequence as shown in FIGS. 1A-1O.
 14. The cleaning composition of claim11 wherein the MP has the amino acid sequence encoding by MP nucleicacid found about 2248 kilobases from the point of origin in Bacillussubtilis.
 15. The cleaning composition of claim 12 wherein the grampositive microorganism is selected from the group consisting of B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, and Bacillus thuringiensis.
 16. A cleaning compositioncomprising a metalloprotease having at least 80% homology with the aminoacid having the sequence shown in FIGS. 1A-1O.
 17. An animal feedcomprising a metalloprotease comprising the amino acid having thesequence shown in FIGS. 1A-1O.
 18. An animal feed comprising ametalloprotease having at least 80% homology with the amino acid shownin FIGS. 1A-1O.
 19. A composition for the treatment of a textilecomprising a metalloprotease comprising the amino acid having thesequence as shown in FIGS. 1A-1O.
 20. A composition for the treatment ofa textile comprising a metalloprotease having at least 80% homology withthe amino acid having the sequence as shown in FIGS. 1A-1O. 21-25.(Cancelled)